The adhesive protein secreted by shells such as hard-shelled mussel (Mytilus coruscus) or acorn barnacle has strong adherent force to a surface of low energy such as a polytetrafluoroethylene surface. One of the amino acids contained in the above mentioned adhesive protein is 4-hydroxyproline.
4-Hydroxyproline is also contained in peptides, such as the peptide of the neurotoxin secreted by the sea snake, which can show physiological effects on the neurons of higher animals. Peptides containing 4-hydroxyproline are, therefore, expected to have utility as a pharmaceutical.
When a peptide is chemically synthesized using an amino acid as a raw material, it first is necessary to use protecting groups to block the functional groups from taking part in a dehydration condensation reaction between the amino and carboxyl groups on the backbone of the amino acid, and the amino, carboxyl and hydroxyl groups on the side chain of the amino acid by protecting groups. The protecting groups then are removed from the intermediates (deprotection) as needed, and allowing the intermediates to take part in the subsequent reaction.
As a method for blocking amino groups on the backbone, protection with benzyloxycarbonyl groups, tert-butyloxycarbonyl groups, Fmoc group or the like is known. Among them, protection with Fmoc groups has been employed because deprotection is feasible under mild basic conditions and chemical synthesis of peptides by a solid-phase technique also is feasible.
Protection of the amino group in amino acids with an Fmoc group and the application of this protection to synthesis of a peptide is disclosed by L. A. Carpino et al. [J. Org. Chem., 37, 3404-3409 (1972)]. Later, J. Meienhofer et al. used it for solid-phase synthesis [Int. J. Peptide Protein Res., 13, 35-42 (1979)].
Techniques such as the symmetric anhydride technique, mixed acid anhydride technique, acid chloride technique, azide technique and active ester technique are known for the dehydration condensation (peptide bonding) of an amino acid whose functional groups (such as amino, carboxyl, and hydroxyl) which do not take part in the dehydration condensation have been protected, and another amino acid whose functional groups (such as amino, carboxyl and hydroxyl) do not take part in the dehydration condensation have been protected. The active ester technique is popular because this technique does not require handling of dangerous reagents such as phosgene. Moreover, the reagents employed in the active ester technique are stable. A variety of reagents for use in the active technique are available on the market.
In the active ester technique, it is known to react electron attractive compounds such as pentafluorophenol or 3-hydroxy-3,4-dihydro- 4-oxobenzo-triazine (hereinafter abbreviated as "Dhbt-OH") represented by formula (IV): ##STR2## in the presence of a coupling reagent such as dicyclohexylcarbodiimide.
The method of using pentafluorophenol as an electron attractive compound was applied to the solid-phase synthesis of a peptide by L. Kisfaludy et al. [Synthesis, 325 (1983); ibid, 303 (1986)]. According to the Kisfaludy et al. teachings, the pentafluorophenyl ester derivative of an amino acid is obtained by protecting the amino groups on its backbone with tert-buthoxycarbonyl or Fmoc groups and then reacting the amino-protected amino acid with equimolar amounts of pentafluorophenol and dicyclohexylcarbodiimide in a solvent such as dioxane or ethyl acetate.
The method of using Dhbt-OH as an electron attractive compound was developed by W. Koenig et al. [Chem. Ber., 103, 2034-2040 (1970)] and was applied to the solid-phase synthesis of a peptide by E. Atherton et al., L. Cameron et al. and P. Goddard et al. [J. Chem. Soc. Chem. Commn., 1763 (1986); ibid, 270 (1987); ibid, 1025 (1988)]. According to those literature articles, the Dhbt ester derivative of an amino acid is obtained by protecting the amino groups on the backbone of the amino acid with tert-buthoxycarbonyl or Fmoc groups and then reacting the amino-protected amino acid with equimolar amounts of Dhbt-OH and dicyclohexylcarbodiimide in tetrahydrofuran.
In addition to the above, active ester methods in which an amino acid derivative of the free carboxyl type is activated in a reaction system and then subjected to coupling, namely the so-called BOP method [B. Castro et al.: Tetrahedron Letters, 1219 (1975)], TBTU method [R. Knorr et al.: Tetrahedron Letters, 30, 1927-1930 (1989)] and PyBOP method [J. Caste et al.: Tetrahedron Letters, 31, 205-208 (1990)] have been developed. In these methods, an activating reagent is added in the presence of a base to a solution containing an amino acid derivative of the free carboxyl type and HOBt to produce a benzotriazole active ester intermediate, followed by a coupling reaction between the intermediate and an amino acid or peptide to be subjected to an extending reaction. Although these methods are believed to have higher reactivity and to cause less side reactions, they are not employed widely.
In addition to the above mentioned methods, introduction of an Fmoc group to the amino group on the backbone of 4-hydroxyproline is known. L. Lapatsanis et al. [Synthesis, 671-673 (1983)].
It is also known to block the hydroxyl group on the side chain of 4-hydroxyproline, serine or threonine with a protecting group such as a benzyl, substituted benzyl and tert-butyl group [E. Wunsch et al.: Chem. Ber., 101, 3659 (1968)].
Chemical synthesis of a 4-hydroxyproline-containing peptide by a combination of the Fmoc method and the active ester method requires either an active ester derivative of 4-hydroxyproline with its amino group protected by an Fmoc group, or an active ester derivative of 4-hyaroxyproline with its amino group protected by an Fmoc group and with its hydroxyl group protected. Such an active ester derivative, however, is not known.